The major health consequences of gonococcal infections occur in the female population. Thus, prevention measures that block or reduce transmission of the gonococcus and other agents that cause sexually transmitted diseases (STDs) would help to improve the overall health of females both in the United States and abroad. Topical microbicides would provide at-risk women a new mechanism to prevent their acquisition of STDs and HIV infection. In keeping with the theme of this Program Project on "Topical Protegrins to Prevent STDs and HIV Infection", we will use Neisseria gonorrhoeae as a model bacterial agent to STDs to study the structural functional aspects of protegrins (a family of porcine-derived antimicrobial peptides) and a newly described antibacterial peptide (LL- 37) that is present in human semen and neutrophils. Protegrins and LL-37 are both cationic peptides that exert broad-spectrum antimicrobial activity in vitro but are structurally distinct. Both peptides also exert potent antigonococcal action in vitro. The susceptibility of gonococci to these peptides appears to be modulated by an efflux pump system (termed mtr) since mutations that result in loss of efflux activity correspond to increased susceptibility of gonococci to both protegrin-1 (PG-1) and LL-37. Genetic and molecular studies indicate that the expression of gonococcal genes (mtrCDE) that encode the mtr efflux pump is subject to both positive and negative transcriptional control processes that could impact the susceptibility of gonococci to antibacterial peptides and other potential topical microbicides in vivo. Additional genes possessed by gonococci are likely to regulate or determine their susceptibility to these compounds. In order to further define the bactericidal capacity of PG-1 and LL-37, we will now use the gonococcus as a model pathogen in assays that determine the action of synthetic variants of PG-1 and LL-37 that have defined amino acid replacements (Specific Aim 1). In addition to its broad-spectrum antimicrobial action, LL-37 also binds bacterial lipopolysaccharide and blocks many of its proinflammatory, damaging activities. The capacity of LL-37 to bind gonococcal lipooligosaccharide (LOS) will be determined since LOS is likely to become available during the action of LL-37 in vivo. The capacity of gonococci to modulate its susceptibility to the lethal action of PG-1 and LL-37 will be studied at the molecular level (Specific Aim 2) in studies that define the mechanism(s) by which the mtr efflux system is regulated. The involvement of additional gonococcal genes in determining its susceptibility to antimicrobial peptides will be ascertained by isolating and characterizing mutants that express deceased susceptibility to PG-1 and LL-37 (Specific Aim 3). The results from this project will help in the development of antibacterial peptides and other agents as topical microbicides to prevent STDs and will advance our knowledge as to how gonococci and other STD pathogens respond to their presence.